Expanded genetic testing of GIST patients identifies high proportion of non-syndromic patients with germline alterations

Traditional genetic testing for patients with gastrointestinal stromal tumors (GISTs) focus on those with syndromic features. To assess whether expanded genetic testing of GIST patients could identify hereditary cancer predisposition, we analyzed matched tumor-germline sequencing results from 103 patients with GISTs over a 6-year period. Germline pathogenic/likely pathogenic (P/LP) variants in GIST-associated genes (SDHA, SDHB, SDHC, NF1, KIT) were identified in 69% of patients with KIT/PDGFRA-wildtype GISTs, 63% of whom did not have any personal or family history of syndromic features. To evaluate the frequency of somatic versus germline variants identified in tumor-only sequencing of GISTs, we analyzed 499 de-identified tumor-normal pairs. P/LP variants in certain genes (e.g., BRCA1/2, SDHB) identified in tumor-only sequencing of GISTs were almost exclusively germline in origin. Our results provide guidance for genetic testing of GIST patients and indicate that germline testing should be offered to all patients with KIT/PDGFRA-wildtype GISTs regardless of their history of syndromic features.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal system 1 . Up to 85-90% of GISTs harbor activating mutations in KIT or PDGFRA and respond to imatinib or sunitinib therapies 2,3 . The remaining 10-15% are KIT/PDGFRA-wildtype and may have somatic loss-offunction (LOF) mutations in succinate dehydrogenase (SDH)complex subunits (SDHA/B/C/D) or NF1, activating mutations in BRAF, FGFR1, or NTRK translocations, or may be driven by germline alterations 1,[3][4][5] . Germline pathogenic variants in SDH-complex genes [6][7][8] , NF1 9 , KIT 10,11 , and PDGFRA 12,13 have been associated with the development of GISTs. Currently, no standard guidelines exist for germline testing in GIST patients. Previous studies on germline contribution to GISTs focused on patients with KIT/ PDGFRA-wildtype tumors, particularly those with syndromic features, and targeted selected genes 4,5,7 , given the assumption that GISTs resulting from germline alterations are frequently syndromic 3,4 . Recent studies, however, demonstrated that syndromic features may be absent in a large proportion of patients with hereditary cancer predisposition 1,14,15 .
Owing to the restrictive pre-selection of patients and genes for genetic testing, the contribution of germline variants to GIST development remains to be fully characterized. To assess whether expanded genetic testing of unselected GIST patients could identify individuals with hereditary predisposition, we analyzed matched tumor-germline sequencing results from 103 patients with GISTs treated at Memorial Sloan Kettering (MSK) Cancer Center (MSKCC) over a 6-year period ( Supplementary Fig. 1). Testing was performed using MSK Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) 16 and included 76-90 cancer predisposition genes (Supplementary Table 1).
Overall, of the 103 patients with GISTs in this cohort, 24 (23%) had a germline pathogenic/likely pathogenic (P/LP) variant in a GIST-associated gene. The cohort of 103 patients included 58 with somatic mutations in KIT, 10 with somatic mutations in PDGFRA, and 35 with KIT/PDGFRA-wildtype GISTs (Fig. 1, Supplementary  Table 2). Strikingly, 69% (24/35) of individuals with a KIT/PDGFRAwildtype GIST harbored a germline P/LP variant in a GISTassociated gene (Fig. 2a, Supplementary Fig. 1). These included 16 patients with SDH-complex gene (seven SDHA, seven SDHB, two SDHC), seven with NF1, and one with KIT germline P/LP variants, accounting for 46%, 20%, and 3% of patients with KIT/ PDGFRA-wildtype GISTs, respectively. Seven (20%) of the patients with KIT/PDGFRA-wildtype GISTs had other somatic driver mutations: four with NF1 biallelic, one with SDHA biallelic LOF, and two with BRAF activating mutations. Three tumors from patients without germline P/LP variants had monoallelic SDHB or SDHD mutations, and due to the limitations in detecting LOH or a second mutation, were considered as inconclusive for somatic mutation status. One patient had neither germline nor somatic mutations identified in GIST-associated genes. SDHC promoter methylation 17 was tested and ruled out in tumors without conclusive driver mutations. Overall, germline P/LP variants in GIST-associated genes were identified in 96% (24/25) of patients with somatic driver-negative tumors.
Germline P/LP variants in cancer predisposition genes not known to be associated with GISTs were identified in eight patients, all of whom had KIT/PDGFRA-mutant GISTs. These included alterations affecting TP53, MLH1, BRCA2, and RECQL in one patient each, as well as two patients with APC p.Ile1307Lys low-penetrance variant and two with monoallelic MUTYH variants (Supplementary Table 2). Loss-of-heterozygosity (LOH) or a second The patient with MLH1 alteration had a microsatellite-stable tumor and homologous recombination deficiency signature was not detected in the patient with BRCA2 alteration, suggesting that these variants likely constitute incidental findings.
Patients with germline GIST-associated variants had a younger median age-of-onset (39.5 vs 52 years, p = 0.01), and were more likely to have metastatic disease (50% vs 20%; p = 0.01) and multifocal lesions (50% vs 18%, p = 0.01) at the time of testing compared to those lacking P/LP variants in these genes (Supplementary Table 3).
Most patients with germline P/LP variants in GIST-associated genes did not present with syndromic features. Of the 16 patients with germline SDH-complex gene defects, only one had syndromic presentation (history of paraganglioma), and another had family history (father with paraganglioma), whereas 88% (14/ 16) had no personal or family history of syndromic features at the time of testing. Thirteen patients had SDHB immunohistochemistry performed, and tumors demonstrated absence of SDHB expression. While 86% (6/7) of patients with germline NF1 alterations had a history of NF1 features, for one patient, identification of the germline NF1 variant led to the recognition of café-au-lait spots and mild axillary/inguinal freckling, consistent with mild neurofibromatosis 15 . Additionally, one (1%) of 103 patients had a pathogenic germline KIT variant (p.Lys509Ile) 18,19 . Overall, 63% (15/24) of patients with germline GIST-associated variants did not have personal or family history of syndromic features, suggesting that a significant proportion of KIT/PDGFRAwildtype GISTs appearing to be sporadic may have underlying germline alterations.
Tumor-only sequencing is commonly performed to assess somatic alterations and may reveal germline variants. To determine the frequency of somatic versus germline variants identified in tumor-only sequencing of GISTs, we analyzed a cohort of de-identified 499 GISTs that received paired tumornormal sequencing using MSK-IMPACT, including tumors from the 103 patients in the initial analysis and 396 patients who did not consent to germline testing ( Supplementary Fig. 1). To mimic a tumor-only sequencing analysis approach, the sequencing data from these tumors was used in an unmatched manner, without subtracting the variant calls detected in the paired normal sequencing data. For distinguishing germline versus somatic variants in the tumor sequencing data, variant allele fraction (VAF) thresholds for predicting germline variants in tumor sequencing recommended by the European Society of Medical Oncology (VAF of >30% for single nucleotide variants (SNVs) and >20% for insertions and deletions (indels)) 20 were applied to P/LP variants identified in the tumors. The matched normal blood sequencing data was used to confirm the germline versus somatic origin of the variants detected in tumors. These VAF thresholds correctly distinguished all true germline and somatic P/LP variants identified in the tumors, as determined by comparison with the  matched normal blood sequencing data. Of the 69 KIT/PDGFRAwildtype GISTs in this cohort, 33 (48%) were found to harbor germline P/LP variants in GIST-associated genes, consistent with our findings in the cohort who received germline testing. P/LP variants identified in tumor-only sequencing of GISTs were almost exclusively germline in certain genes, such as BRCA1, BRCA2 (4/4 variants), and SDHB (10/11 variants). Conversely, P/LP variants in genes such as KIT (430/431 variants) and RB1 (9/9 variants) were primarily somatic (Fig. 2b). These observations can help identify the subset of GIST patients to offer germline testing based on tumor-only sequencing results.
This study has limitations. Detailed clinical assessments could only be performed for 103 patients who consented to germline testing. Additionally, limitations exist in detecting certain variants such as structural variants or low-level mosaicism.
Despite these limitations, our results support that germline testing should be offered to all patients with KIT/PDGFRA-wildtype GISTs, regardless of their history of syndromic features, and should target all GIST-associated genes at minimum. In our cohort of 103 patients with GISTs who consented to germline analysis, 23% had a germline P/LP variant in a GIST-associated gene, and an additional 8% had a germline P/LP variant in an additional cancer susceptibility gene, suggesting that all GIST patients, regardless of their tumor testing status, could benefit from germline testing. Germline alterations in GIST-associated genes may also confer high risk for other cancers, such as paragangliomas and pheochromocytomas for SDH-complex genes and nerve sheath tumors, breast cancer, and gliomas for NF1. Therefore, their identification has critical implications for future cancer surveillance and clinical management of the patients, as well as appropriate care of their at-risk family members. Determining the somatic and germline alterations underlining tumor development is also critical for appropriate targeted therapy selections, as SDHdeficient and NF1-related tumors are known to respond poorly to traditional imatinib therapy and differentiating the different genetic alterations in KIT/PDGFRA-wildtype GISTs can guide treatment choices 3,4 . Finally, our analysis on 499 tumor-normal pairs suggests that the identification of P/LP variants in certain genes in tumor-only sequencing of GISTs may indicate germline testing. Additional large-scale studies on the phenotypic spectrum of GIST-associated genes will help in developing clinical guidelines for expanded genetic testing in GIST patients.

Patient cohort
The de-identified cohort consisted of tumor-normal pairs from 499 consecutive patients with GIST who were treated at MSKCC and had MSK-IMPACT (ClinicalTrials.gov identifier, NCT01775072) paired tumor-blood DNA sequencing test 21,22 between April 2015 and June 2021. Germline analysis cohort consisted of 103 patients with GIST, who were a subset of the larger cohort and prospectively consented to germline analysis as part of MSK-IMPACT. Patients were ascertained through their treating physicians and referral was at the discretion of the physicians. The presence/absence of personal/family history of syndromic features were determined based on review of pre-testing and post-testing clinical geneticist and oncologist physical examination and family history assessments that included inquiry about features related to genetic disorders associated with the development of GISTs. All patients provided written informed consent for testing under a Memorial Sloan Kettering Cancer Center Institutional Review Board (IRB)-approved protocol (IRB#12-245).

MSK-IMPACT testing
MSK-IMPACT is a New York State Department of Health approved assay and was performed in our CLIA-approved laboratory. Nextgeneration sequencing was performed on DNA isolated from matched blood and tumor specimens, as described 16,[21][22][23] . DNA fragments were captured using custom-designed biotinylated probes (NimbleGen) and sequenced on an Illumina HiSeq 2500 as paired-end 100 bp reads 21,22 25 . Copy number variants (deletions and duplications of single or multiple exons) were detected and analyzed using an in-house developed pipeline 16,22,23 , which identifies copy number aberrations by comparing sequence coverage of targeted regions to standard diploid normal. Variants were filtered using 25% (for SNVs) and 15% (for indels) variant allele fraction and 20× coverage thresholds. All variants with <1% population frequency in the Genome Aggregation Database (gnomAD) 26 were reviewed and classified by clinical molecular geneticists and molecular genetic pathologists based on the American College of Medical Genetics and Genomics (ACMG) guidelines 27 .
LOH, microsatellite instability (MSI), and mutation signature analyses LOH was determined using segmented allele-specific copy number calls from FACETS tool 28 , an open-source software that utilizes aligned sequence bam files from next-generation sequencing and performs analysis for joint segmentation of total-and allele-specific read counts and integer copy number calls corrected for tumor purity, ploidy and clonal heterogeneity to estimate LOH. Segments with a minor allele copy number of 0 were classified as having LOH. The allele undergoing LOH was determined based on the VAF of the germline variants in the tumor. MSI status of the tumors were assessed using clinically validated MSIsensor program 29 , which computes length distributions of microsatellites per designated regions in paired tumor and normal sequence data and identifies the percentage of microsatellite loci that are unstable in the tumor genome as compared to its matched normal. Evidence of MSI at ≥10% of analyzed loci was considered as MSI-high, ≥3 to <10% was considered as indeterminate MSI status, and <3% were considered as microsatellite stable. Tumor mutation signatures were determined based on MSK-IMPACT data by assigning the mutations in each sample to constituent mutation signatures from a set of 30 signatures described previously 23,30,31 .

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article. D Mandelker et al.